habitat segregation and biochemical activities of marine ...habitat segregation of marine...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1985, p. 781-7900099-2240/85/100781-10$02.00/0Copyright © 1985, American Society for Microbiology

Habitat Segregation and Biochemical Activities of Marine Membersof the Family VibrionaceaeUSIO SIMIDU* AND KUMIKO TSUKAMOTO

Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan

Received 18 March 1985/Accepted 2 July 1985

A comparative study of marine members of the family Vibrionaceae with the technique of numericaltaxonomy revealed habitat segregation as well as a cosmopolitan nature of species distribution among thevibrios in different marine environments. The bacterial strains analyzed were isolated from seawater,sediments, phyto- and zooplankton, and fish in the Indian Ocean, the South and East China Sea, and WestPacific Ocean, and coastal areas of Japan. A total of 155 morphological, physiological, and biochemical testswere carried out for each of 405 strains examined. The results showed that most of the large taxonomicalclusters which emerged from the computation corresponded to ecological groups which have particular niches.For instance, each group of seawater vibrios inhabited a particular water layer of limited depth range, in spiteof the fact that strains of the group were isolated from sampling locations spread over a wide area from theIndian Ocean to Japanese coast. Various vibrio groups showed remarkable differences in their physiologicaland biochemical activities, and the activities of each group seemed to correspond with its ecological niche. Thestrains which inhabited surface-water layers grew fast and actively utilized many high-molecular-weightorganic compounds and carbohydrates that are derived from fresh, easily degradable organic matter presentin the surface waters, whereas the middle- and deep-water vibrios did not decompose most of thehigh-molecular-weight organic compounds except chitin but, rather, utilized some carbohydrates and organicacids which seemed to be derived from refractory particulate organic matter present in the deeper waters.

The members of the family Vibrionaceae comprise one ofthe predominant bacterial groups in marine environments.They constitute a considerable part of marine heterotrophicbacterial populations. In general, members of the familyVibrionaceae contribute 10 to 50% of the heterotrophicbacteria from coastal seawater samples that grow on ordi-nary agar media used in marine bacteriology, although thereare some exceptions among samples from highly eutrophic-ated areas (10, 17, 20). They are also abundant in pelagicseawaters of the Pacific and Indian Oceans and in the SouthChina Sea (21). In marine environments, members of thefamily Vibrionaceae are closely associated with many kindsof marine animals from plankton (18, 24) to fish (13, 19, 26).Their symbiotic relationship with luminous fish has beenknown since the last century (7). Two species of the familyVibrionaceae, Vibrio anguillarum and V. parahaemolyticus,are pathogens for marine animals, and several species arealso known as human pathogens. Other than the well-knownV. cholerae and V. parahaemolyticus, an increasing numberof species such as V. vulnificus and V. fluvialis are nowrecognized as the causative agents of human disease (3). Thepresent status of the taxonomy of the family Vibrionaceae iswell documented by West and Colwell (25). In Bergey'sManual ofSystematic Bacteriology, Baumann and Schubert(2) list 20 species for the genus Vibrio, 4 species for the genusAeromonas, 3 species for the genus Photobacterium, and 1species for the genus Plesiomonas.During the last 15 years we have isolated strains of the

family Vibrionaceae from various marine environments,including seawater of the Pacific and the Indian Oceans, andthe East and South China Sea, from various depths up to2,000 meters, and also from phyto- and zooplankton and fish.In the present study, the method of numerical taxonomy was

used to examine a large number of strains, mostly derived

* Corresponding author.

781

from oceanic marine environments that are relatively lowtemperature and oligotrophic in nature.

MATERIALS AND METHODS

Bacterial strains. A total of 387 strains were examined. Thetype cultures used in this study were as follows: Aeromonasharveyi NCMB 2, A. hydrophyla NCMB 89, A. ichthyosomaNCMB 86, A. liquefaciens NCMB 87, A. salmonicida ATCC14174, Lucibacterium harveyi NCMB 42 and NCMB 1280,Photobacterium mandapamensis NCMB 391 and NCMB1198, P. phosphoreum NCMB 844, Plesiomonas shigelloidesNCIB 9242, Pseudomonas fluorescens NCMB 3756, P.formicans NCMB 23, Vibrio alginolyticus NCMB 1903 andATCC 17749, V. anguillarum NCMB 6 and NCMB 829, V.costicola NCMB 788, V. costicolus NCMB 701, V. fischeriNCMB 1281, V. ichthyodermis NCMB 407, V. marinusATCC 15382, V. pierantonii NCMB 25, and V. para-haemolyticus NCMB 1902 and ATCC 17802. The sources ofthe other strains are given in Table 1. The strains includedgram-negative, carbohydrate-fermenting bacteria isolatedfrom seawater and marine animals. The seawater strains werecollected from various water layers at several samplingstations in the Indian Ocean (stations 13 and 15 at KH-76-5cruise), the South China Sea (stations 17, 19, 20, and 21 atKH-76-5 cruise), the East China Sea (station 22 at KH-76-5cruise and station 29 at KH-75-1 cruise), the Pacific Ocean(stations 24 and 28 at KH-75-1 cruise), and coastal areas

around Japan, namely Suruga Bay (station U-2 at KT-74-7cruise), Sagami Bay (station A at KT-79-2 cruise), andOtsuchi Bay (stations 4 and 8). The number of strains selectedfrom each water layer is given in Table 1. Special care wastaken to ensure a random selection of strains from eachsampling depth and location, although the number of selectedstrains was not proportional to the vertical distribution ofvibrios. Some gram-negative, curved rods, which were notcarbohydrate fermenting and hence were not members of the

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782 SIMIDU AND TSUKAMOTO

TABLE 1. Numbers and sources of the strains included in the numerical taxonomy study

No. of isolates from the following locations:

Source Indian South East Pacific Japanese NorthernOcean China China Ocean coast Pacific Total

Sea Sea Ocean

Seawater0 m 7 11 1 5 21 7 5210 m 8 2 1020-30 m 7 9 6 3 3 7 3540-50 m 5 13 2 19 39100-125 m 7 10 6 2 25200 m 6 7 3 7 23400 m 7 7 4 2 20600-800 m 8 3 1 1 131,000-1,200 m 6 7 1 141,600-2,000 m 4 4

Sediment 3 4 5 1 13Plankton

Phytoplankton 13 13Zooplankton 11 15 26

FishIntestine 18 15 14 47Gill 6 6Skin 4 1 5

Diseased (moribund) fish and shellfish 17 17

family Vibrionaceae, were used for a comparative study. Toassess experimental errors, 18 reference cultures wereselected at random from the whole set of strains used. Beforetesting, each culture was examined for purity by streaking onagar medium (ORI medium given below).

Media. Stock cultures were maintained on ORI shakemedium and stored at 20°C. The ORI medium contained 1.0g of proteose peptone no. 3 (Difco Laboratories), 1.0 g ofyeast extract (Difco) 0.5 g of phytone (BBL MicrobiologySystems), 0.2 g of sodium thiosulfate, 0.05 g of sodiumsulfite, and 0.04 g of ferric citrate dissolved in a mixture of900 ml of aged seawater and 100 ml of distilled water. Unlessspecified otherwise, ORI medium was used as the basalmedium for the physiological and biochemical tests.Morphology. Colonial morphology and pigmentation were

determined on ORI agar plates after incubation for 7 days at200C.Luminescence on ORI agar medium was observed after 1

and 3 days of incubation. The presence of fluorescentpigment was detected on King medium (12). Micro-morphology was examined with heat-fixed smears of 24-hcultures after staining with 0.2% fuchsin solution. Gramstaining was carried out with the Hucker modification of thegram stain (4). The presence of pleomorphism, curved andstraight rods, chains, filaments, tapered ends, and cellulargranules was recorded. The stained preparation was photo-graphed, and the cell size was measured on photographicfilms under a microscope at low magnification. The produc-tion of poly-f3-hydroxybutyrate was tested on a seawater(75%) medium containing 0.5% sodium-,-hydroxybutyrateand 0.05% yeast extract. The presence of granules in thecells was observed after 2 and 4 days incubation at 200C.Motility of the strains was determined microscopically witha phase-contrast microscope.

Physiological and biochemical tests. The ability to grow at2, 5, 10, 26, 34, and 40°C was determined on ORI agarmedium. The dependency of growth on pH was examined onORI agar medium by adjusting the pH to 4.0, 5.0, 6.0, 8.0,9.0, and 10.0. The ability to grow at NaCl concentrations of

0, 0.2, 0.5, 1.0, 2.0, 5.0, 7.0 and 10.0% was determined onORI agar medium supplemented with appropriate amountsof NaCl. The test for growth rate was carried out with ORIbroth. A portion of a 24-h culture on the broth was inocu-lated on fresh medium and incubated at 20°C, and the opticaldensity at 660 nm was read after 20 and 48 h. The slope of theincrement of optical density between the initial time andeach incubation time was taken as a measure of growth rate.

Susceptibility to antibiotics was tested with the diskdiffusion method (BBL Sensi-System). The following antibi-otics were tested: ampicillin (10 ,ug), carbenicillin (100 ,ug),cephalothin (30 ,ug), chloramphenicol (30 ,ug), clindamycin (2,ug), colistin (10 ,ug), erythromycin (15 ,ug), gentamicin (10,ug), kanamycin (30 ,ug), penicillin (10 U), and streptomycin(10 ,ug). For the carbohydrate metabolism test and someother biochemical tests, the Miniteck System (BBL) wasextensively used. The following tests were carried out withthe system: acid production from arabinose, cellobiose,dextrose, galactose, lactose, levulose, maltose, mannose,melibiose, raffinose, sucrose, trehalose, xylose, adonitol,dulcitol, glycerol, inositol, manitol, and sorbitol; hydrolysisof esculin, starch, and urea; utilization of citrate and malon-ate; nitrate reduction, Voges-Proskauer test, argininedihydrolase, lysine decarboxylase, o-nitrophenyl-,B-D-galactopyranoside test, phenylalanine deamination, utiliza-tion of citrate and malonate, and indole production. Produc-tion of H2S was determined with both the Minitek systemand in SIM medium (Eiken Chemical Co. Ltd., Tokyo,Japan). The production of gas from carbohydrates wastested in ORI broth containing 0.5% carbohydrates. InvertedDurham tubes were inserted to detect gas production. Utili-zation of amino acids and organic acids was determiend withan artificial seawater medium, which is composed of 2.33%NaCl, 0.26% K2SO4, 0.074% MgSO4 - 7H20, 0.018% CaC12,0.014% KH2PO4, 0.002% ferric citrate, 0.01% EDTA, and0.01% yeast extract and has a pH of 7.5. To the medium forthe test of utilization of organic acids, 0.013% (NH4)2SO4was added. The following amino acids and organic acidswere added at a concentration of 0.1%: alanine, leucine,

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HABITAT SEGREGATION OF MARINE VIBRIONACEAE 783

NO OFPHENON STRAINS90 100

-~ Para 11-= = 23 S51 4

S2 28

- ~ Algi 8

@ j Phos 6_ $ ] Mand 11

~~~~~~~~~ Fl 4

3 ; S F2 7-E S3 17

S4 11ag.. F3 6

S 7

P1 12

P2 7

- ~ S6 10

_ S7 30

- ": Harv 4

S8 14

S9 39

S_ 7

~~~~~~~~ P3 8

Sil 10

.~~~~~~~~ F4 699

P4 7

~~~~~~~ F6 4.=_. S13 3

FS14 3

FIG. 1. Simplified dendrogram of strains examined in the present study based on simple matching coefficients and unweighted averagelinkage.

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TABLE 2. Numbers and sources of the strains in main phenaNo. of isolates from the following sources:

SeawaterPhenon

Indian South East West apaneseWNorth Fish Plankton Type TotalOndian China China Pacific coast OaceanulurOcean Sea Sea Ocean Ocean

PARA 1 1 1 6 2 11Si 2 1 1 4S2 8 10 4 6 28ALGI 1 5 2 8PHOS 1 1 1 2 1 6MAND 1 3 1 5 1 11Fl 4 4F2 7 7S3 4 7 1 3 2 17S4 7 4 11F3 6 (3)' 6S5 1 2 4 7P1 2 1 4 5 12P2 1 2 4 7S6 5 2 1 1 1 10S7 5 11 1 4 7 2 30HARV 1 1 1 1 4S8 2 12b 14S9 1 20 2 10 7 40S1o 1 3 3 7P3 1 1 6 8Sil 3 2 1 1 3 10F4 1 5 6F5 2 2 5 (5) 9P4 3 4 7F6 4 4S13 3 3S14 2 1 3

a Numbers within parenthesis show the strains from fish caught in the northern area.b Includes two strains from sediments of the North Pacific Ocean and Otsuchi Bay.

threonine, methionine, phenylalanine, proline, glutamicacid, lysine, histidine, arginine, asparagine, formate, ace-tate, n-valerate, lactate, d-tartarate, malonate, glutamate,azelaate, citrate, oxalate, and d-gluconate. Gelatin hydroly-sis was tested in ORI medium containing 25% gelatin.Hydrolysis of casein was determined on an agar mediumcomposed of 0.1% proteose peptone no. 3, 0.1% yeastextract, 0.05% phytone, and 2.0% Bacto-Agar (Difco). Tothe melted medium, an additional one-fifth, by volume, ofautoclaved skim milk solution (10%) was added and plated.The presence of DNase was detected by the method ofJeffries et al. (8). The hydrolysis of Tween 60, Tween 80, andtributirin was determined by the Rhodes method (15). Chitindigestion was detected on ORI agar plates containing resus-pended chitin particles (22).Computer analysis. The test results were coded 0 for

negative results, 1 for positive results, and 9 for uncompar-able or missing data. Tests that were positive or negative inall cases were deleted from the data matrix. Reference dataobtained from 18 pairs of the same strains were processedwith a FACOM M180IIAD (HSA) (Fujitsu Ltd., Tokyo,Japan) computer, and the tests that were believed to behighly subjective, namely, granules in the cells, intermediatecell size, and utilization of some organic acids, were deletedfrom the data matrix. Finally, 159 features representing 128tests were computed. Similarity among strains was calcu-lated using the simple matching coefficient. Clustering wascarried out by single linkage and unweighted average linkagemethods (25). The program used is described elsewhere (9).

RESULTS

Phena. Most of the paired strains, which were tested atdifferent times during the present study, were combined intotaxa at a similarity coefficient of 87 to 95% (mean, 92%)when the unweighted average linkage method was adopted.There was 1 exception out of the 18 pairs, where the pair(strains no. 135 and 414) joined into a cluster at a similaritylevel of 81%. This pair has a similarity coefficient of 86.3%when compared with each other. Similarly, some pairs oftype cultures of the same species (L. harveyi, V.parahaemoliticus, V. anguillulum, and V. alginolyticus)joined into taxa at a similarity coefficient of 91 to 93%.However, pairs of P. mandapamensis and V. costicola (V.costicolus) were divided into remote phena; the similarityvalues of these pairs were 80 and 84%, respectively. Strainsthat were not capable of fermenting carbohydrates (eightstrains from seawater and two type cultures of P.fluorescens) joined Vibrio strains at a similarity coefficient of67 to 84% when the average linkage method was employed.The distance between strains of the same species was closerwith the average linkage method than with the single linkagemethod. These results suggest that, with the clustering of thepresent study, the level of similarity which distinguishesspecies may be set at approximately 80 to 85% when theaverage linkage method is used.At a similarity coefficient of 86 to 90%, the 405 strains

examined in this study clustered into 58 groups when theunweighted average linkage method was used. A simplified



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FIG. 2. Distribution pattern of seawater vibrios in different water layers. The area in the figure does not represent vertical quantitativedistribution of a phenon (see the text).

dendrogram is shown in Fig. 1. Most of the single-strainphena were omitted from the dendrogram, and clusters thatwere composed of more than two strains are shown. Therewere 28 distinct clusters that included more than threestrains.

Sources of the strains in the clusters are given in Table 2.Most of the strains isolated from fish were clustered together(phena Fl, F2, F3, F4, F6) and did not include strains fromseawater and plankton, although there were some clusterswhich included seawater strains and fish strains (phenaPARA, MAND, S7, Sli, and F5). Likewise, most strainsderived from plankton were concentrated into six phena (S2,P1, P2, S9, P3, and P4), although in some of these phenaplankton strains were mixed with seawater strains.A striking feature is that the clusters of seawater strains

were composed of vibrio groups that inhabit particular waterlayers and are segregated vertically from each other. Thus,phena S2 and S9 were composed of strains isolated fromsurface-water layers at depths of 0 to 100 m, whereas phenaS4 and Sli included strains isolated from layers at depths of400 to 1,2Q0 m. Strains of clusters S3, S6, S7, and S10 wereisolated from seawater samples collected between the layersthat were inhabited by the surface and deep-water groups.Thus, habitat segregation was observed among the differentgroups clustered by taxonomic criteria.The habitat segregation that was observed was apparantly

independent of the area of sampling (Table 2). For example,phenon S2, whose members inhabit surface-water layers (0to 100 m), consisted of isolates from the Indian Ocean, theSouth China Sea, and the Pacific Ocean as well as fromplankton. Similarly, the inhabitants of middle-water layers(phenon S3) included isolates from diverse areas from theIndian Ocean to the Japanese coast. Apparently, waterdepth is a more critical factor than geographic location indetermining the distribution of species. There are, however,

some differences in geographic distribution among thegroups that inhabit water layers of the same or similar depth.Among luminescent bacteria isolated from the marine

environment, segregation was also observed. One group ofluminescent bacteria was included in phena PHOS andMAND, comprising isolates from fish intestine and seawater(O to 800 m) as well as type cultures of P. phosphoreum(NCMB 1198) and P. mandapamensis (NCMB 844), whereasthe other group of luminescent bacteria fell into phena P1and P2, which included cultures from plankton and surfaceseawater.

Figure 2 shows the distribution of clusters in various waterlayers. The total number of strains at each sampling depthwas corrected so as to be the same throughout the wholewater column from the Indian Ocean to the Japanese coast.Hence, the area for each cluster in the figure is not propor-tional to the number of viable vibrios, which decreases withincreasing depth. Although there are considerable fluctua-tions in the pattern of decrease depending on the time andplace of sampling, generally vibrio counts were reduced by 1order of magnitude from the surface to 200 m and by 1further order of magnitude to 1,000 m. Taking account of thisdecrease in number, Fig. 2 gives an approximate image ofthe distribution of various vibrio groups in the sea.

Several phenotypic characteristics of these clusters areshown in Tables 3 and 4.

DISCUSSIONIn the present study 354 strains of vibrios and related

organisms from pelagic marine environments were examinedalong with 25 type cultures and 8 nonvibrio strains, whichserved as reference strains. Most of the isolates from pelagicseawater could not be assigned to known representatives'ofthe family Vibrionaceae examined in this study. In Bergey'sManual of Systematic Bacteriology, Bauman and Schubert

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TABLE 3. Selected characteristics of main phena

% of isolates with indicated characteristic

Characteristic PARA ALGI PHOS MAND(P )a Si (4) S2 (28) (8) (6) (11) Fl (4) F2 (7) S3 (17) S4 (11) F3 (6) S5 (7) P1 (12)

MorphologyWidth >0.42 ,um 8 0 57 27 100Width >0.50 pum 0 0 10 0 83Length/width ratio >2.2 100 100 97 100 0Length/width ratio >5.0 33 80 0 18 0Coccoids to cocci 8 0 50 9 100Curvature (slight) 83 80 17 55 50Curvature (intensive) 0 20 0 0 0

Motility 100 100 100 100 100Luminescence 0 0 0 0 33Growth

20C 0 0 0 0 10050C 0 0 0 18 100340C 100 80 80 100 0400C 58 80 60 100 0pH 6.0 8 0 0 36 67pH 10.0 8 40 80 27 00.2% NaCl 8 0 0 18 00.5% NaCl 83 80 97 100 837.0% NaCl 42 0 0 100 0

Growth rates0.12 OD increment/24 100 100 100 100 17h-0.24 GD increment/24 100 100 97 100 0hc0.36 OD increment/24 83 80 60 100 0h

PHBb accumulationArginine dihydrolaseLysine decarboxylaseNitrate reductionVoges-Proskauer testIndole productionPhosphataseDegradation of:

EsculinAlginic acidCaseinChitinDNAGelatinLaminaranStarchTween 80

Acid from:CellobioseGalactoseGlycerolMaltoseMannitolMannoseSorbitolSucroseTrehaloseXylose

Gas from:GlucoseMannose

Utilization of:AcetateCitrateMalonateTartarateAlanineArginineHistidineLeucineLysinePhnylalanineProline

17 0 10 0 170 20 0 0 17

67 0 10 100 83100 100 97 100 1000 0 0 100 67

100 100 97 91 0100 100 100 100 100

75 40 87 91 050 0 0 18 090 100 100 100 0100 100 100 50100 100 100 100 100100 100 100 100 00 0 0 0 0

100 100 100 100 1792 100 100 73 33

92 100 100 82 092 0 3 73 100100 100 93 100 10075 100 100 100 100100 20 20 100 0100 60 37 64 100

8 0 0 0 042 20 0 91 0100 100 100 100 0

0 0 0 0 0

83 80 2975 40 050 80 1000 0 0

83 40 2942 62 1008 20 100

100 67 10050 0 0

40 2964 67 57100 0 017 0 092 20 00 0 00 0 0

100 40 1000 0 0

82 20 29

82 100 100 5753 90 71 076 0 14 1000 0 0 082 100 100 2941 0 14 860 0 0 29

100 82 100 1000 0 0 0

65 100 076 100 0 8624 0 100 436 0 57 0

76 0 100 00 0 0 00 0 0 0

76 0 100 00 0 0 0

100 0 83 100

8342830

83500

1008

00

75800

178

36

27 0 0 35 0 0 29 27

0 0 0 0 0 0 0 9

9 75 860 20 0

25 40 0100 100 7142 80 00 0 0

92 20 86

0 0 570 0 08 0 0

100 20 2967 80 4325 0 08 0 08 0 43

33 100 0

0 0 14100 100 092 60 140 100 1000 0 29

100 100 710 0 08 0 430 0 570 0 0

41 45 86 290 0 0 012 0 0 076 82 100 10035 0 100 00 0 0 0

100 27 57 100

100 0 0 140 0 0 0

65 9 0 7165 100 86 100100 27 100 100100 73 14 4324 0 0 06 6 43 57

100 100 100 100

100 9 100 0100 100 100 059 9 100 088 0 100 570 0 0 0

100 100 100 430 0 0 0

47 0 0 053 27 71 57100 100 0 0

0 0 0 0 100 0 100 0 0 0 0 00 0 0 0 100 8 100 0 6 0 29 0

50 20 13 91 6725 0 63 55 025 0 57 36 067 100 37 100 6792 100 97 100 83100 100 100 100 0100 100 93 100 075 60 43 100 042 40 20 64 058 0 13 55 092 100 97 100 83

36 40 00 0 00 0 0

82 60 8691 0 8627 60 719 0 00 0 29

27 0 00 0 0

100 60 86

35 0 0 00 0 0 00 0 0 14

94 0 57 71100 91 43 10088 100 57 10082 73 0 8618 9 0 296 27 14 430 0 0 094 100 100 100

4200

250

83100

00

2510010092017

100

0800000000

00

000

75100100018330

91

786


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TABLE 3-Continued

% of isolates with indicated characteristicCharactenistic PARA Si (4) S2 (28) ALGI PHOS MAND Fl (4) F2 (7) S3 (17) S4 (11) F3 (6) S5 (7) P1(12)

Susceptibility to:Ampicillin 0 0 7 0 17 27 80 86 6 82 14 43 50Carbenicillin 0 0 0 0 0 0 60 43 0 27 0 0 33Cephalothin 33 20 7 9 67 27 80 86 47 55 14 71 75Colistin 0 0 0 0 100 100 100 100 100 91 0 14 0Gentamicin 25 80 50 27 100 55 40 43 94 82 71 71 92Penicillin 0 0 0 0 0 0 0 14 0 0 0 0 8

a Numbers within parentheses indicate the number of strains in a phenon.b PHB, Poly-,B-hydroxybutyrate.

(2) listed 20 Vibrio species, 13 species of which were notincluded in the present study. Comparison of the maincharacteristics of the clusters that emerged in the presentstudy with the species listed in Bergey's Manual ofSystem-atic Bacteriology revealed that most of the clusters ofseawater vibrios did not fall into the listed species, with theone eKception that the majority of luminescent bacteria wereclosely related to P. phosphoreum, P. mandapamensis, orV. fisheri. Strains of phenon S2 were similar to V. campbeliiin biochemical characteristics, although most of them wereslightly curved rods, and in this respect that differed from V.campbellii. Kaper et al. (11) carried out a numerical taxo-nomic study on the vibrios from estuarine environments andshowed that most of the isolates belonged to known species.Most of the known vibrio species were not psychrophilic.There are only two known species, V. logei and V. marinus,which definitely grow at 4°C. On the other hand most of themiddle- and deep-water strains in this study grew well at 2°C.Further taxonomic and molecular genetic investigations arerequired to determine taxonomic relationships among theoceanic vibrios and between the known species and thesestrains.A remarkable feature derived from the results is that the

clustering of marine vibrios by the technique of numericaltaxonomy revealed habitat segregation in the vibrio popula-tion. Some cluster (phena S2 and S9) contained isolatesalmost exclusively from surface water and plankton, in spiteof the fact that they had been isolated from remote areasranging from the Indian Ocean to the Japanese coastalregion. Other clusters (phena S3, S6, and S10) were inhab-itants of subsurface waters, and still other clusters (S4 andSli) were those of further deeper waters. In most of theclusters the strains were isolates from remote samplingareas, which showed the cosmopolitan nature of speciesdistribution of marine bacteria. The growth temperature andsalinity range of each phenon appeared to reflect environ-mental conditions of the phenon habitat. Most of the strainsin phenon S2 and S9, which are isolates from surface waters,did not grow at temperatures lower than 5°C, but they didtolerate lower concentrations of NaCl, whereas most strainsin phena S4 and S1l, isolates from deeper waters, grew at50C. None of the deep-water vibrios grew at .340C, whereas58 to 80% of the surface vibrios did. More remarkabledifferences were observed in the biochemical activities ofisolates from different water layers. The surface vibrios(phena S2 and S9) showed much greater metabolic activitythan the vibrios of deeper-water layers (phena S4 and Sl1).Strains of phena S2 and S9 grew much faster than thedeep-water vibrios. They actively decomposed high-molecular-weight organic compounds such as starch,esculin, gelatin, casein, and DNA and produced phospha-

tase. On the other hand, deep-water vibrios did not hydro-lyze starch, casein, and esculin, and the percentage ofphosphatase producers was low.

Clear contrasts were also observed in the pattern ofcarbohydrate utilization. Almost all of the strains of surfacevibrios produced acid from maltose, cellobiose, trehalose,and glycerol, and none of them decomposed galactose andxylose. All of the strains of phenon S9 and 37% of phenon S2strains produced acid from mannitol. On the other hand,most of the deep-water vibrios produced acid from galactoseand mannose, but did not decompose mannitol, cellobiose,and trehalose. Strains of phenon S4 also produced acid fromxylose. Strains of phena S3, S6, S7, and S10, which aremainly composed of isolates from the intermediate layersbetween the surface and deep waters, showed characteris-tics intermediate in all respects between the surface- anddeep-water vibrios. They showed moderate growth rates andan intermediate range of growing temperature and pH. Alldecomposed gelatin and DNA, and some of them decom-posed esculin and casein. The pattern of carbohydrateutilization was also intermediate between that of the surface-and deep-water vibrios. On the other hand, the percentage ofchitin decomposers was lower than those of both surface-and deep-water vibrios, and some strains decomposedlaminaran, which is not accessible to surface- and deep-water vibrio groups.The different biochemical activities observed in the vari-

ous taxonomic clusters seem to reflect the nutritional envi-ronment in which the strains of each cluster live. The surfaceseawater layers contain more fresh and easily decomposableorganic substances derived from phyto- and zooplanktonand bacteria. These organic substances are quickly decom-posed and utilized by bacteria and zooplankton at thesurface. Riley (15) estimated that about 90% of organicmatter produced in the surface water is decomposed beforeit reaches 100 m of depth. Thus, most particulate organicmatter is decomposed by bacteria and other organismsbefore it reaches the deep-water layers. Handa andTominaga (5) showed that not only was particulate organicmatter less abundant in deeper water, but also it had a highercarbon/nitrogen ratio than that of surface waters, whichindicates that the particulate organic matter in deep watercontains a smaller amount of proteinaceous material. Thus,easily decomposable nutrients are depleted in the deeperwater layers, and bacteria living there have to utilize eithermore refractory organic compounds and their decompositionproducts or smaller amounts of dispersed organic com-pounds derived from zooplankton and bacteria. The bacteriain phena S4 and S11 did not hydrolyze proteins and starch,but most of the strains actively utilized galactose, mannose,and xylose (phenon S4), which are formed through the

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TABLE 4. Selected characteristics of main phena% of isolates with indicated characteristic

Characteristic HARVP2 (7)a 56 (10) 57 (30) (4)V 58 (14) S9 (40) S10 (7) P3 (8) 511 (10) F4 (6) F5 (9) P4 (7) F6 (4)

MorphologyWidth >0.42 ,umWidth >0.50 LmLength/width ratio >2.2Length/width ratio >5.0Coccoids to cocciCurvature (slight)Curvature (intensive)

MotilityLuminescensGrowth

20CS°C340C400CpH 6.0pH 10.00.2% NaCl0.5% NaCl7.0% NaCl

Growth rate<0.12 OD increment/24 hrc0.24 OD increment/24 hrc0.36 OD ihcrement/24 hr

PHBb accumulationArginine dihydrolaseLysine decarboxylaseNitrate reductionVoges-Proskauer testIndole productionPhosphataseDegradation of:

EsculinAlginic acidCaseinChitinDNAGelatinLaminaranStarchTween 80

Acid from:CellobioseGalactoseGlycerolMaltoseMannitolMannoseSorbitolSucroseTrehaloseXylose

Gas from:GlucoseMannose

Utilization of:AcetateCitrateMalonateTartarateAlanineArginineHistidine

LeucineLysinePhenylalanineProline

50 7513 25

100 10033 08 58

83 580 0

100 1000 0

0 670 75

100 1000 80 500 00 00 750 0

100 9288 3313 013 420 0

33 3288 330 0

100 0100 100

38 1000 0

88 83100 58100 10075 1000 17

100 0100 100

50 1000 100

13 2525 670 00 1000 00 0

88 330 67

81 0 71 59 7129 0 43 10 2971 100 50 66 570 0 0 0 074 25 64 90 10058 100 64 83 710 50 29 5 0

100 75 100 100 10050 0 0 0 0

81 0 79 5 1493 0 85 32 1480 25 8 59 433 0 0 12 0

90 0 0 2 00 0 23 17 860 0 0 0 0

81 25 64 95 860 0 14 5 14

100 75 93 100 10040 0 57 100 1003 0 14 90 57

67 100 14 5 430 0 0 0 00 0 0 0 090 75 100 100 10032 0 0 2 00 100 100 90 100

100 100 100 100 86

13 100 100 98 1000 25 71 0 14

45 100 93 100 8677 100 86 100 100100 100 100 100 10084 100 100 100 10032 25 29 0 010 100 93 95 100

100 100 100 100 100

3 100 93 98 8694 75 93 0 8619 75 93 98 8642 100 100 98 1000 25 100 93 100

97 75 100 5 860 0 0 0 016 0 36 0 068 100 100 100 10077 0 0 2 0

0 1000 91

100 4538 00 82

100 36100 0100 1009 0

0 550 75

75 013 00 00 00 0

38 550 0

88 038 013 013 670 180 18

63 730 64

13 0100 64

0 013 0

100 038 9100 8275 200 0

25 0100 27

0 00 730 550 1000 00 1000 00 90 00 0

57 78 22 5029 67 22 1386 100 100 500 11 0 0

71 11 44 50100 100 89 7557 44 11 0100 100 100 1000 0 0 13

0 0 00 33 0 00 33 67 750 0 0 00 0 0 00 0 0 250 0 0 00 22 0 1000 0 0 0

0 11 78 1000 0 0 00 0 0 0

100 56 33 1000 0 0 0o0 0 0 0

100 56 56 1000 0 0 00 0 22 0

83 89 89 100

43 0 22 250 0 0 00 0 63 014 44 22 10086 67 100 1000 44 89 750 0 22 1000 0 56 500 67 100 100

0 0 0 00 0 0 00 33 44 014 0 22 00 0 22 0

43 50 11 1000 0 0 00 0 0 00 22 67 00 0 0 0

0 0 3 0 0 0 0 0 18 0 0 0 00 0 10 0 0 0 0 0 36 0 11 0 0

0 500 0

25 00 83

88 10075 6763 420 17

13 013 083 100

65 0 0 10 03 0 7 54 140 0 0 39 43

97 75 36 24 29100 75 100 93 8673 100 100 90 10043 100 100 100 867 0 57 41 573 0 57 20 290 50 0 2 0

100 100 100 95 100

0 1050 050 0100 20100 45100 9100 038 063 00 0

100 27

0 11 0 00 0 0 00 0 0 0

71 78 78 5043 67 100 7550 78 100 1000 0 89 1000 11 22 00 22 22 500 0 0 25

83 89 100 100



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HABITAT SEGREGATION OF MARINE VIBRIONACEAE

TABLE 4-Continued

% of isolates with indicated characteristicCharacteristic HARV

P2 (7)- S6 (10) S7 (30) (4) S8 (14) S9 (40) S10 (7) P3 (8) S11 (10) F4 (6) F5 (9) P4 (7) F6 (4)

Susceptibility to:Ampicillin 0 0 7 0 17 27 80 86 6 82 14 43 50Carbenicillin 0 0 0 0 0 0 60 43 0 27 0 0 33Cephalothin 33 20 7 9 67 27 80 86 47 55 14 71 75Colistin 0 0 0 0 100 100 100 100 100 91 0 14 0Gentamicin 25 80 50 27 100 55 40 43 94 82 71 71 92Penicillin 0 0 0 0 0 0 0 14 0 0 0 0 8

a Numbers within parentheses indicate the number of strains in a phenon.b PHB, Poly-,B-hydroxybutyrate.

decomposition of refractile organic compounds such as agar,carrageenan, mannan, and xylan. Handa et al. (6) showedthat detrital material from deeper (500-m) water containsmuch higher amount of mannose, xylose, arabinose, andgalactose in its hot water-extractable fraction than that fromsurface (10-m) water. If we assume that vibrios in deeperwaters depend for their nutrients on the particulate organicmatter falling from the surface, their data are well in accordwith the ability of deep-water vibrios to utilize these sugars.The surface vibrios did not utilize these sugars, whereas theycan utilize other sugars like cellobiose, glycerol, andmaltose, which are not accessible to the deep-water vibrios,and which are produced during the process of decompositionof phytoplankton by bacteria or digestion by marine animals.The concept of habitat segregation has been well estab-

lished in ecology since Imanish first proposed it in 1949. Itimplies temporal or spatial segregation of the habitats of twoor more species of a related group due to differences in thebiotic and abiotic environment. The present study revealedhabitat segregation among clusters of marine members of thefamily Vibrionaceae defined on purely taxonomic grounds,which indicates that a particular taxonomic group (species orgenus) may correspond to or coincide with an ecologicalgroup having a particular niche and function in a givenecosystem.There exists a possibility that the bacterial groups ob-

served in the middle- and deep-water layers do not representthe indigenous bacterial flora in a given water layer, but aretransient bacteria attached to falling particulate organicmatter. Some types of particulate organic matter, notablyfecal pellets of large zooplankton and nekton, are known todescend fairly rapidly at a rate of 40 to 100 m per day (1, 16),and Fukami (K. Fukami, Ph.D. thesis, University of Tokyo,1982) suggested that there is a vertical succession of bacte-rial flora associated with the process of decomposition ofparticulate organic matter as it falls through the watercolumn.However, the fact that the phena S2 and S9 excluded all

middle-water vibrios, with the exception of four strains inphenon S9, which contained isolates from 1,200 m, and thatthe middle- and deep-water flora will not grow under thetemperature and salinity conditions of surface waters,strongly suggests that the clusters that emerged from thepresent study represent indigenous vibrio flora that haveadapted to environmental conditions of the depth at whichthey were sampled.Some clusters were split into two groups, one inhabiting

the surface- to middle-water layers and the other inhabitingthe deeper, 1,200- and 1,600-m layers. Three strains out offour deep-water isolates of phenon S9 did not grow at the in

situ temperature of 5°C and may have been transporteddown from the surface.

Strains from the coastal regions of the Northern PacificOcean formed clusters different from those of strains fromsouthern areas. Strains from fish and plankton also, ingeneral, comprised distinct clusters. From these results weconclude that taxonomic groupings emerging from numericaltaxonomy coincide with ecological groupings of a specificecological niche.

ACKNOWLEDGMENT

We thank R. R. Colwell, University of Maryland, for her valuablesuggestion throughout the present study and for reading themanuscript. Y. Ezura, Hokkaido University, kindly provided us withthe collection of strains from fish and seawater in the Northern Pacificregions. Thanks are also due to the captains and crews of R/VHakuho-Maru and Tansei-Maru, for helping with the sampling.The present study was partly supported by Japan-U.S. coopera-

tive research project no. IPS-MBR 004 and by a Grant for ScientificResearch from the Ministry of Education of Japan (project no.55613).

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