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INTERNATIONAL OURNAL OF SYSTEMATICACTERIOLOGY,ct. 1992, p. 621-627Copyright 1992, International Union of Microbiological Societies0020-7713/92/040621-07 02.00/0

Vol. 42, No. 4

Alteromonas atlantica SQ nov. and Alteromonas carrageenovorasp. nov., Bacteria Tha t D ecompo se Algal PolysaccharidesMASAYO AKAGAWA-MATSUSHITA,* MASARU MATSUO,* YOSUKE KOGA,l

AND KAZUHIDE YAMASAT03Department of Chemistry, Universityof Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu807 nstitute of Applied Biochemistry, The Universityof Tsukuba, Tsukuba-shi, Ibaraki 305, andInstitute of Applied Microbiology, The Universityof Tokyo, Bunkyo-ku, okyo 113, Japan

We studied seven strains of aero bic, m arine, polarly flagellated bacteria which decompose alginate, agar,and carrageenan. The major respiratory quinone of these strains was ubiqu inone-8. The G + C content of theirDNA was 39.5 to 41.7 mol . Pseudomonas atlantica IAM 12927 and the conspecific five isolates wereconcluded to constitute a single species distinguished from the other nonpigmented Afteromonas species byDNA-DNA hybridization (homology values o f more than 82 ) and phenotypic similarity (similarity coeffi-cients, based on assimilation of 145 carbon compounds, were 79 to 96 ). Pseudomonas carrageenovora IAM12662, the sole extant strain, was distinct from P. atlantica and other AZteromonas species in DNA-DNAhybridization and phenotypic features. T axonomic affinity to Afteromonas espejiana was indicated byDNA-DNA hybridization with P atlantica IAM 12927 and the five conspecific isolates (39 to 55 ) and withP. carrageenovora I M12662 (43 to 45 ). Phenotypically, higher similarity values (79 to 89 ) forassimilation of 145 carbon compounds were shared between A. espejiana IAM 12927 T and the six conspecificstrains, including P. atlantica IAM 12927. Alteromonas atlantica sp. nov. (type strain, I M12927, =ATCC19262, =NCIMB 301) and Afteromonuscarrageenovora (type strain, I M12662, =IF0 12985, =ATCC 43555,=NCIMB 302) are proposed for P. atlantica I M 12927 and the conspecific five isolates and P.carrageenovora I M12662, respe ctively. A set of phenotypic features which differen tiate the twoAfteromonasspecies is described.

Marine algal polysaccharides are potent resources forbiomass, and their microbial degradation is primarily neces-sary for the development of their practical use. The enzymesthat degrade polysaccharides of diversified structures are ofinterest from an enzymological standpoint. Biomacromole-cule-degrading microorganisms were found in Vibrio-relatedbacteria (4,6, 7, lo , yellow Cytophaga-likebacteria (16,21,30), aerobic Pseudomonas-like bacteria (5, 13, 17, 23, 26-28), and some actinomycetes (10). The genus Alteromonaswas created in 1972 for marine bacteria that are Pseudomo-nas like but which have a lower G+C content (8). rRNA-DNA hybridization experiments showed that the genusAlteromonas comprises two phylogenetic descents (22).Some marine strains, formerly identified as Pseudomonasspecies, belonged in the Alteromonas haloplanktis rRNAbranch (9, 22).Pseudomonas atlantica LAM 12927, which was orig-inally isolated by W. Yaphe (27), is an agar-decomposingstrain, and its agarase is widely used and commerciallyavailable. Pseudomonas can-ageenovora IAM 12662 is acarrageenan-decomposing strain, and several enzymologicalstudies of its carrageenases have been reported (12, 14, 24,25, 28, 29). The names of these two strains have not beenvalidated, and their generic assignment has been thought tobe incorrect. They belong in the genusAlteromonas, accord-ing to rRNA-DNA hybridization by De Vos et al. (9).However, their assignment at a species-specific level hasremained unclarifizd. M. Matsuo isolated several strains ofgram-negative, polarly flagellated rods which decomposedcarrageenan from seaweed collected in Japan. In this study,DNA-DNA hybridization and phenotypic characterization,including carbon source assimilation tests and chemotaxo-

* Corresponding author.

nomic analyses, were performed to clarify the taxonomicassignment of five unnamed strains, P. atlantica IAM12927, and P arrageenovora I M12662, all of whichdecompose agar and/or carrageenan.MATERIALS AND METHODS

Bacterial strains. The strains used in this study are listed inTable 1.Five strains of Alteromonas atlantica were isolatedfrom seaweeds collected at Inubousaki, Choshi-city, ChibaPrefecture, Japan. To enrich carrageenan-decomposing bac-teria, aliquots of 10 ml of seawater containing 200 mg of apowdered brown alga Lessonia species) were inoculatedwith pieces of seaweed and incubated with reciprocal shak-ing (160 times per minute) at 25C for 66 to 70 h. Thedecomposition of carrageenan in culture broth was checkedby estimating the amount of reducing sugar produced andmonitoring the decrease of viscosity of the culture fluid. Thecarrageenan-decomposing strains were isolated from theenriched cultures by plating on agar plates. The medium wascomposed of 2.5 g of Casamino Acids (catalog no. 1230;Difco), 2.5 g of carrageenan, 50 ml of 0.2 M sodium phos-phate buffer (pH 8.0), 500 ml of seawater, 450 ml of distilledwater, and 15 g of agar; its pH was adjusted to 7.0 beforeautoclaving. Colonies were picked up after 3 days of incu-bation at 25C. A. atlantica I M12927 was received asPseudomonas atlantica NCMB 301 (NCIMB 301 atpresent), which was originally isolated by W. Yaphe in 1957(27) as an agar-decomposing pseudomonad. A. can-ageeno-vora IAM 12662 was received as P. can-ageenovora IF012985. Type strains of nonpigmented Alteromonas species,including A. macleodii L M 12920T, A. espejiana IAM12640T,A. haloplanktis IAM 12915T,A. undina IAM 12922=,A. ni yaciens IAM 13010T, and A. tetraodonis IAM14160 were used as reference strains (superscript T de-

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622 AKAGAWA-MATSUSHITA ET AL. INT. J. SYST. ACTERIOL.TABLE 1. DNA-DNA homology among Alteromonas strains

Strain% Association with 3H-labeled DNA from:G+C content(mol%) A. atlanticd A. espejiana A. carrageenovora A. tetraodonisIAM 12927T IAM 12640T IAM 12622T IAM 14160T

A. atlantica IAM 12927TaA. atlantica 512-MA. atlantica 512-TA. atlantica 512-WA. atlantica 511-TA. atlantica T9A. espejiana IAM 12640TA. carrageenovora IAM 12662TbA. haloplanktis IAM 12915TA. undina IAM 12922TA. nigrifaciens IAM 13010TA. tetraodonis IAM 14160TA. macleodii IAM 12920TM . communis IAM 12914TM. vaga IAM 12923=E. coli IAM 1264

41.240.841.040.641.740.741.439.541.640.140.640.846.247.048.451.3

1009595100828243212725326444

403941554753100432325217543

26

451002121234443

23

2226

100

4=NCIMB 301, =ATCC 19262. (NCIMB is the National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland; ATCC is the American TypeCulture Collection, Rockville, Md.; IAM is the Institute of Applied Microbiology, The University of Tokyo, Tokyo, Japan.)=IF0 12985, =NCIMB 302, =ATCC 43555. ( IF0 is the Institute for Fermentation Osaka, Osaka, Japan.)=Strain K-12.

notes the type strain). The type strains of Marinomonasvaga and Marinomonas communis (IAM 12923* and IAM12914T) and Escherichia coli IAM 1264 (strain K-12) wereused as distantly related reference strains.Morphological, biochemical, an d p hysiological characteri-zation. Conventional phenotypic characteristics (Table 2)were determined by methods we have described previously(1,3). Alginate hydrolysis activity was determined by bothour previously described method (1) and the method thatwas described by Schlesner et al. (18). In the latter method,the formation of a clear zone was detected by flooding theplate of marine agar 2216 (catalog no. 0979; Difco) containing1 sodium alginate with a diluted Lugol solution whichproduces brown alginate-iodine complex. Simple matchingcoefficient determination was used to indicate phenotypicsimilarities on the basis of results of assimilation tests with145 organic carbon substrates as sole sources of carbon (3).The auxanographic API galleries for 48 carbohydrates (API50CH galleries), 49 organic acids (API 50AO galleries), and49 amino acids and amines (API 50AA galleries) (Labora-toire de Recherche API, La Balme-les-Grottes, Montalleu-Vercieu, France) were used with the defined basal mediumdescribed by Baumann et al. (8). The test with 2-ketogluco-nate was omitted because precipitate appeared in fresh

TABLE 2. Ubiquinone compositions of the Alteromonasstrains usedStrain Ubiquinone compositionQ-7 Q-8 Q-9 Q-10

A. atlantica IAM 12927=' T 96 2A. atlantica 512-M 1 96 1 tA. atlantica 512-T T 95 2 tA. atlantica 512-W T 97 t tA. atlantica 511-T 1 97 2A. atlantica T 9 T 96 2A. carrageenovora IAM 12662Tb T 97 1 TComposition expressed as percent total ubiquinone. T, between 0.5 and1.0%; t, less than 0.5%.Data from reference 2.

medium without inoculum. Positive results were recordedwhen better growth than what was without any carboncompound was obtained.Determination of moles percent G+C content and DNA-DNA hybridization. Methods for isolation and purification ofDNA were described previously (1).Moles percent G+Ccontents of DNA were determined by the high-performanceliquid chromatography (HPLC) method (20). DNA-DNAhybridization experiments were performed by the membranefilter method (19). Reference DNAs were labeled with[3H]dCTPby nick translation (15)by using a nick translationkit (Takara Shuzou Co., Ltd., Kyoto, Japan). The reassoci-ation mixture consisted of 10 pg of unlabeled single-strandedDNA immobilized on a nitrocellulose membrane (typeTM-3; pore size, 0.3 pm; Toyo Roshi Kaisha, Ltd., Tokyo,Japan) and 0.1 pg of labeled reference DNA suspended in0.1% sodium dodecyl sulfate-2x SSC (0.3 M NaCl plus 0.03M trisodium citrate). After incubation for 40 h at 62 C, thefilters were washed and dried according to the methoddescribed by Suzuki et al. (19). The radioactivity of the filterwas measured with a model LS 7000 liquid scintillationcounter (Beckman Instruments, Inc., Fullerton, Calif.).Quantification of DNAs was based on hyperchromicity asdescribed by Johnson (11).

Respiratory quinone analysis. Quinones were extractedfrom lyophilized cells harvested at early stationary growthphase with chloroform-methanol (2:l) and were purified bythin-layer chromatography on a silica gel with benzene as adeveloping solvent. The molecular species of quinone wasdetermined from its &value, and HPLCwas employed toanalyze the composition of isoprenologs (1, 2). Ubiquinoneis abbreviated as Q, and the number of the side-chainisoprenoid units is shown by the number after the hyphen.RESULTS

Five newly isolated strains, P. atlantica IAM 12927,and P. carrageenovora IAM 12662 were polarly flagel-lated, gram-negative, nonfermentative rods (Fig. 1). Noperitrichous cells were observed when the strains werecultivated on solidified media. Their G+C contents were


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VOL.42, 1992 A. ATLANTICA SP. NOV., A. CARRAGEENOVORA SP. NOV. 623

FIG. 1. E lectron micrographs of the nega tively stained cells ofA. atlantica and A. carrageenovorudem onstrating their polar flagellation.(A)A. atlantica IAM 12927T. (B )A . atlantica 512-M (IAM 14161). (C)A . atlantica T9 (IAM 14165). D)A. carrageenovoruIAM 12662=. Barsrepresent 1 pm.39.5 to 41.7 mol% (Table l), uggesting that they should beassigned to the genus Alteromonas (8). Their major ubiqui-none was 0 - 8 (at least 95% of the total, Table 2), accompa-nied with small amounts of Q-9, Q-7, and Q-lo. The profile isessentially the same as that of Alteromonas species (2).DNA-DNA hybridization results (Table 1)showed that thefive newly isolated strains were closely related to Patlantica IAM 12927, with homology values from 82 to100%. Phenotypically, 79 to 96% similarity resulted from 145carbon substrate assimilation tests among these six closelyrelated strains as shown in Fig. 2. They were classified asbelonging in a single species. Differential biochemical andphysiological features and the carbon source assimilationprofiles are shown in Table 3. All strains tested were positivefor the following features: acid production from glucose;growth at pH 5.5 and 8.5; Na+ requirement; hydrolyses ofesculin, Tween 80, gelatin, casein, and DNA; tyrosinedecomposition; phosphatase; H2Sproduction; growth in thepresence of vibriostatic agent OD29 (2,4-diamino-6,7-diiso-propylpteridine, 150 pg per disk); oxidase; catalase; meth-ylene blue reduction; and assimilation of acetate, propio-

nate, pelargonate, caprate, oxalate, glucose, esculin,maltose, starch, and L-glutamate. All strains tested werenegative for the following features: growth at pH 5.0; deni-trification; production of fluorescein, pyocyanin, and prodi-giosin; xanthine decomposition; indole formation; 3-keto-lactose production from lactose; growth with 0.4%phenethyl alcohol; tryptophan deaminase; phenylalaninedeaminase; methyl red test; and assimilation of malonate,glutarate, DL-glycerate, L-tartrate, citraconate, itaconate,adipate, pimerate, suberate, azerate, sebacate, glycolate,D-tartrate, meso-tartrate, phenylacetate, benzoate, m-hy-droxybenzoate, D-mandelate, L-mandelate, phthalate, isoph-thalate, tert-phthalate, glycine, D-a-alanine, L-a-alanine,.L-leucine, L-isoleucine, L-norleucine, L-valine, DL-norval-ine, ~~-2-aminobutyrate,-serine, L-threonine, L-cysteine,L-methionine, L-histidine, D-tryptophan, L-tryptophan, trig-onelline, L-aspartate, L-citrulline, DL-kynurenine, L-proline,betaine, creatine, 9-alanine, ~~-3-arninobutyrate,~ - 4 - a m i -nobutyrate, ~~-5-aminovalerate,-amino-benzoate, 3-ami-nobenzoate, 4-aminobenzoate, urea, acetamide, sarcosine,ethylamine, butylamine, amylamine, ethanolamine, 1,4-di-


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624 AKAGAWA-MATSUSHITA ET AL INT. J. SYST.BACTERIOL.

A atlantica IAM 12927

- atlantica 512-M

A atlantica 512-T 8 9 8 7-A atlantica 512-W 8 7 8 7 9 6-- atlantica 511-T 7 9 8 3 8 9 8 9

A atlantica T9 8 7 8 3 8 9 8 9 8 9

- espejiana IAM 12640r 8 3 7 9 8 9 8 9 8 9 8 5

- carrageenovora IAM 12662= 8 1 6 6 7 2 7 4 6 4 7 2

4 haloplanktis IAM 12915T 6 4 6 4 7 7 6 6 6 6

A undina IAM 12922r 6 6 6 2 5 8 5 8 6- -- tetraodonis IAM 14160f 5 5 5 5 6 4 6 0 6 5 3

- nigrifaciens IAM 13010 6 6 5 8 7 2 7 2 7 2 6 4

- macleodii IAM 12920T 4 7 4 3 5 7 5 7 5 7 5 3FIG. 2. Similarity coefficients (in percents) am ong Alteromonas strains, based on the results of assimilation of 145 carbon compounds.

aminobutane, benzylamine, spermine, histamine, trypt-amine, D-arabinose, L-xylose, p-methyl+-xyloside, sor-bose, erythritol, adonitol, inositol, sorbitol, xylitol, dulcitol,a-methyl-D-mannoside, inulin, D-lyxose, D-tagatose, D-fu-cose, L-fucose, D-arabitol, and L-arabitol.A . espejiana L4M 12640T proved to have values of pheno-typic similarity to the six strains (79 to 89%) nearly the sameas those among the six strains, though its DNA homologyvalues were distinctly lower (39 to 5 5 ) . However, theformer six strains are clearly distinguished fromA. espejianaby the following tests: agar hydrolysis, the citrate utilizationtest, and assimilation of D-xylose and D-mannose as shown inTable 2. They are also phenotypically differentiated fromother known nonpigmentedAlteromonas species.DISCUSSION

On the basis of the above-mentioned phenotypic andgenotypic distinctness, we propose A . atlantica sp. nov. tobe the classification for the six agar-decomposing strains,including Pseudomonas atlantica I M12927. A . atlan-tica should be considered to be a member of A . haloplanktisrRNA branch because of its significant DNA homology withA . espejiana, which was classified on this branch (22). Patlantica was originally described by Humm (10) in 1946.Humm isolated and characterized two strains from Atlanticcoastal areas, but both have been lost. A. atlantica I M12927 was isolated and partially characterized as P. atlan-tica by Yaphe in 1955 (28) and has been sometimes referredto as the type strain. We propose strain IAM 12927 be thetype strain of A. atlantica.

The distinctness of P. cawageenovora I M12662 wasshown by the hybridization experiments (Table 1)and therelatively low phenotypic similarity (below 81%; Fig. 2) toany of the tested strains. The highest DNA homology value,45%, was obtained when the DNA was hybridized with thatof A. espejiana. This indicates no more than a genericrelationship to A . espejiana, which can be distinguished bymeans of tests for starch hydrolysis, agar hydrolysis, citrateutilization, and assimilation of L-malate and aconitate (Table2). We propose Alteromonas cawageenovora as the namefor this enzymologically important, carrageenan-decompos-ing strain, IAM 12662 (type strain). Differential characteris-tics of other nonpigmented Alteromonas species are pre-sented in Table 2.Description of A. atluntica sp. nov. A . atlantica (at. lan.ti. ca.Gr.adj. atlantica, of the Atlantic [Ocean]). Gram-neg-ative, strictly aerobic, polarly flagellated bacterium isolatedfrom seaweeds. Cells are rod shaped, with rounded ends,and are 0.5 to 0.8 pm in diameter and 1.5 to 2.3 pm longwhen the organism is grown on marine agar 2216 medium(catalog no. 0979; Difco); the cells occur singly or in pairs.No endospores or capsules are formed. Peritrichous flagel-lation is not observed when the organism is cultivated onsolidified media. Colonies on marine agar 2216 medium arebeige to pale yellow orange, are smooth and convex withentire edge, and grow to a diameter of 7.0 to 10.0 mm in 5days at 25C. The colonies on agar media were stronglydepressed by agarase activity. Sodium ion is essential forgrowth. Are mesophilic and neutrophilic chemoorgano-

trophs which grow at temperatures between 5 and 35C andat pHs between 5.5 and 8.5. They never grow at 40C and pH


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VOL. 42, 1992 A. A T W I C A SP. NOV., A CARRAGEENOVORA SP. NOV. 625TABLE 3. Differential phenotypic features of tested Alteromonas strains

Result for strain:Feature A. espejiana A. halo- A. undina A. nigr i - A. mucleo- A. tetra-

I M12662T I M12640T IAM 12915T 12922T 13010T 12920T 14160Tplankth I M f a c k m I M d i i W o&nh L M. atianti- A. atiantica A* carra-caa IM 12927~ geenovora

Acid from mannitolGrowth at:5C35C40CpH 9.0Nitrate reductionMelaninlike dark pigmentONPG testHydrolysis ofStarchAlginateAgarCarrageenanUrease2-Ketogluconate from gluconateLitmus milk coagulationCi rate utilizationLevan productionArginine hydrolysisAssimilation of:ButyrateIsobutyrateti-ValerateIsovaleraten-CaproateHeptanoateCaprylateSuccina eMaleateFumarateDL-Lacta eDL-3-HydroxybutyrateD-MalateL-MalateLevulinate2-KetoglutarateMesaconateAconi a eCitrateo-Hydroxybenzoatep-HydroxybenzoateL-PhenylalanineL-TyrosineL-OrnithineL-LysineL-ArginineD-GlucosamineGlycerolL-ArabinoseD-RiboseGalactoseFructoseD-MannoseRhamnoseMannitola-Methyl-D-glucosideN -Acetyl-D-glucosamineAmygdalinArbutinSalicinCellobioseLactoseMelibiose

D-Xylose

10010010008317331001001001008317010006775678383831750336705001700001710010033001700500

8300010010010001000010000100100

100

+-

--

-

-

+-

--

------

--

-

-

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626 AKAGAWA-MATSUSHITA ET AL.TABLE 3-Continued

INT. J. SYST.BACTERIOL.

Result for strain:Feature A. espejiana A. halo- A. undina A. ni&i- A. macleo- A. tetra-

IAM 12662T I M12640T IAM 129 UT 12922T 13OlOT 12920T 14160Tplanktis I M faciens IAM diiIAM odonis IAM. atlanti- A. atlantica A. cam-c IAM 12927T geenovom

SucroseTrehaloseMelezitoseRaffinoseGlycogenGentiobioseD-TuranoseGluconate5-Ke oglucona ePercent positive strains. For all tests except arginine hydrolysis, n = 6; for arginine hydrolysis, n = 4.

5.0. All strains except IAM 12927T grow at pH 9.0. Acid isproduced from glucose and mannitol. Are positive for hy-drolyses of esculin, Tween 80, gelatin, casein, starch, DNA,alginate, and agar. Chitinase is not produced. Are positivefor oxidase and catalase; in the o-nitrophenyl-P-D-galacto-pyranoside (ONPG) test; and for tyrosine decomposition,phosphatase, H,S production, litmus milk coagulation, andmethylene blue reduction. Are negative for nitrate reduc-tion, denitrification, cell pigmentation, amidase, xanthinedecomposition, indole production, 2-ketogluconate produc-tion from gluconate, 3-ketolactose production from lactose,citrate utilization, 0/129 sensitivity, growth on mediumcontaining 0.4% phenethyl alcohol, and deaminases fortryptophan and phenylalanine; are negative in the methyl redtest; and are negative for curdlanlike polysaccharide produc-tion. Some strains produce carrageenase, urease, acetyl-methylcarbinol, and melaninlike dark pigment on tyrosine-containing medium. Luminescence is never observed.Acetate, propionate, butyrate, isobutyrate, n-valerate, iso-valerate, pelargonate, caprate, oxalate, succinate, aconitate,citrate, glycerol, galactose, glucose, fructose, D-mannose,mannitol, amygdalin, esculin, cellobiose, maltose, lactose,melibiose, sucrose, trehalose, starch, glycogen, gentiobiose,and L-glutamate are utilized by 67% or more of the strains assole sources of carbon and energy. The following com-pounds are not utilized by at least 67% of the strains:n-caproate, caprylate, malonate, maleate, glutarate, adipate,pimerate, suberate, azerate, sebacate, glycolate, DL-lactate,DL-glycerate, ~~-3-hydroxybutyrate, -malate, L-malate,D-t ar r at e L-t ar r ate, meso- artra te levu ina e, 2-ke oglu-tarate, citraconate, itaconate, mesaconate, phenylacetate,benzoate, o-hydroxybenzoate, m-hydroxybenzoate, p-hy-droxybenzoate, D-mandelate, L-mandelate, phthalate, isoph-thalate, tea-phthalate, glycine, D-a-alanine, L-a-alanine,L-leucine, L-isoleucine, L-norleucine, L-valine, DL-norva-line, ~~-2-aminobutyra te,-serine, L-threonine, L-cysteine,L-methionine, L-phenylalanine, L-tyrosine, L-histidine,D-tryptophan, L-tryptophan, trigonelline, L-aspartate, L-glu-tamate, L-ornithine, L-lysine, L-citrulline, DL-kynurenine,L-proline, betaine, creatine, p-alanine, ~~-3-aminobutyate ,~~-4-aminobutyrate,~-5-aminovarelate,-aminobenzoate,3-aminobenzoate, 4-aminobenzoate, urea, acetoamide, sar-cosine, ethylamine, butylamine, amylamine, ethanolamine,benzylamine, 1,4-diaminobutane, spermine, histamine, trypt-amine, D-glucosamine, erythritol, D-arabinose, L-arabinose,D-ribose, D-xylose, L-xylose, adonitol, p-methyb-xyloside,sorbose, rhamnose, dulcitol, inositol, sorbitol, a-methyl-D-mannoside, a-methyl-D-glucoside,N-acetyl-D-glucosamine,

arbutin, salicine, inulin, melezitose, raffinose, xylitol,D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D-ar-abitol, L-arabitol, gluconate, and 5-ketogluconate. The G+Ccontent of the DNA ranges from 40.6 to 41.7 mol%. Iso-prenoid quinone is Q-8. Strains have been isolated frommarine macroalgae collected from both the Atlantic andPacific coastal areas. The type strain is I M 12927(=NCIMB 301, =ATCC 19262). Strains 512-M, 512-T,512-W, 511-T, and T9 were deposited to the Institute ofApplied Microbiology culture collection as IAM 14161, I M14162, IAM 14163, IAM 14164, and IAM 14165, respec-tively.Description of A. carrageenovora sp. nov. A. carrageeno-vora (car.ra.gee. no'. vo. ra. M.L.n. carrageenum, carra-geen, another name for carrageenan; L.v. voro, to devour;M.L.fem.adj . carrageenovora, carrageenan decomposing).Gram-negative, strictly aerobic, polarly flagellated bacte-rium isolated from seaweeds. Cells are rod shaped, withrounded ends, and are 0.7 to 0.8 pm in diameter and 1.9 to2.5 pm long when the organism is grown on marine agar 2216medium (catalog no. 0979; Difco); the cells occur singly or inpairs. No endospores or capsules are formed. Peritrichousflagellation is not observed when the organism is cultivatedon solidified media. Colonies on marine agar 2216 mediumare beige to pale yellow orange, circular, and smooth andconvex with entire edge and grow to a diameter of 6.0 to 8.0mm in 5 days at 25C. Sodium ion is essential for growth. Aremesophilic and neutrophilic chemoorganotrophs which growat temperatures between 5 and 35C and at pHs between 5.5and 9.0. No growth occurs at 40C and pH 5.0. Acid isproduced from glucose and mannitol. Are positive for hy-drolyses of esculin, Tween 80, gelatin, casein, DNA, algi-nate, and carrageenan. Chitin, starch, and agar are nothydrolyzed. Are positive for oxidase and catalase; in theONPG test; and for tyrosine decomposition, phosphatase,urease, arginine hydrolysis, citrate utilization, H2S produc-tion, litmus milk coagulation, and methylene blue reduction.Are negative for nitrate reduction, denitrification, cell pig-mentation, amidase production, xanthine decomposition,indole production, 2-ketogluconate production from glu-conate, 3-ketolactose production from lactose, 0/129 sensi-tivity, growth on medium containing 0.4% phenethyl alco-hol, and deaminases for tryptophan and phenylalanine; arenegative in the methyl red test; and are negative for curd-lanlike polysaccharide production. Melaninlike diffusibledark pigment was produced on tyrosine-containing medium.Luminescence was never observed. Acetate, propionate,n-valerate, caprylate, pelargonate, caprate, oxalate, succi-


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VOL.42, 1992 A. ATLANTICA SP. NOV., A. CXRRAGEENOVOM SP. NOV. 627nate, fumarate, L-malate, 2-ketoglutarate, citrate, 0-hydrox-ybenzoate, p-hydroxybenzoate, L-glutamate, glycerol, ga-lactose, glucose, fructose, mannitol, amygdalin, esculin,cellobiose, maltose, lactose, melibiose, sucrose, raffinose,starch, and gentiobiose are utilized as sole sources of carbonand energy. The following compounds are not utilized:butyrate, isobutyrate, isovalerate, n-caproate, heptanoate,malonate, maleate, glutarate, adipate, pimerate, suberate,azera e, sebaca e, glycola e, DL-lactate, DL-glycer a e, D L - ~ -hydroxybutyrate, D-malate, D-tartrate, L-tartrate, rneso-tar-trate, levulinate, citraconate, itaconate, mesaconate, aconi-tate, phenylacetate, benzoate, rn-hydroxybenzoate, D-man-delate, L-mandelate, phthalate, isophthalate, tert-phthalate,glycine, D-a-alanine, L-a-alanine, L-leucine, L-isoleucine,L-norleucine, L-valine, DL-norvaline, ~~-2-aminobutyrate,L-serine, L-threonine, L-cysteine, L-methionine, L-phenylala-nine, L-tyrosine, L-histidine, D-tryptophan, L-tryptophan,trigonelline, L-aspart ate, L-ornithine, L-lysine, L-citrulline,L-arginine, DL-kynurenine, L-proline, betaine, creatine, 9-al-anine, ~~-3-aminobutyate,~-4-aminobutyrate,~-5-amino-varelate, 2-aminobenzoate, 3-aminobenzoate, 4-aminoben-zoate, urea, acetoamide, sarcosine, ethylamine, butylamine,amylamine e hanolamine, benzylamine, 1,4-diaminobu anespermine, histamine, tryptamine, D-glucosamine, erythritol,D-arabinose, L-arabinose, D-ribose, D-xylose, L-xylose,adonitol, P-methyl-D-xyloside, D-mannose, sorbose, rham-nose, dulcitol, inositol, sorbitol, a-methyl-D-mannoside,amethyl-D-glumside, N-acetyl-D-glucosamine, arbutin, sali-cine, trehalose, inulin melezitose, glycogen, xylitol, D-tura-nose, D-lyxose, D-tagatose, D-fumse, L-fucose, D-arabitol,L-arabitol, gluconate, and 5ketogluconate. The G+C contentof the DNA is 39.5 mol%. Isoprenoid quinone is Q-8. The typestrain is IAM 12662 =IF0 12985, =NCIMB 302, =ATCC43555).

ACKNOWLEDGMENTSWe thank Aiko Hirata (Institute of Applied Microbiology, TheUniversity of Tokyo) and Mitsuru Yokoyama (University of Occu-pational and Environmental Health) for their technical advice onelectron microscopy.

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