System. Appl. Microbiol. 25, 360–375 (2002)© Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

A Detailed Phenotypic Characterisation of the Type Strainsof Halomonas Species

JUAN ANTONIO MATA, JOSÉ MARTÍNEZ-CÁNOVAS, EMILIA QUESADA, and VICTORIA BÉJAR

Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, University of Granada, Spain

Received: June 12, 2002

Summary

We have made a detailed phenotypic characterisation of the type strains of 21 species within the genusHalomonas and have also studied any possible intraspecific variation of strains within H. eurihalina, H.halophila, H. maura and H. salina. We used 234 morphological, physiological, biochemical, nutritionaland antimicrobial susceptibility tests. Nutritional assays were carried out using both classical and minia-turized (BIOLOG system) identification methods. Two different numerical analyses were made using theTAXAN program; the first included the differential data from all the tests carried out whilst the secondused only the 57 tests with the highest diagnostic scores (≥ 0.5). The results of both analyses were quitesimilar and demonstrated the phenotypic heterogeneity of the Halomonas species in question. At a 62%similarity level the type species were grouped into three phena, the main difference between them beingthe capacity of those included within phenon A (H. aquamarina, H. meridiana, H. cupida, H. pantelle-riensis and H. halmophila) to produce acids from sugars. The species grouped in phenon C (H. camp-isalis, H. desiderata and H. subglasciescola) used fewer organic substrates than the others. The remainingstrains were included in phenon B. H. marisflavi was clearly distinct and thus was not included in any ofthe three phena. High phenotypic similarity (more than 88%) was found between Halomonas campisalisand Halomonas desiderata.The results of our work should allow researchers to minimise the tests required to arrive at a reliablephenotypic characterisation of Halomonas isolates and to select those of most use to differentiateHalomonas species from each other.

Key words: Halomonas – halophilic bacteria – phenotypic characterisation – BIOLOG – numericaltaxonomy

Introduction

Extremophiles are microorganisms that grow underconditions hostile to most organisms (HORIKOSHI, 1997).Some of them, such as bacteria which thrive in hyper-saline environments, have been recognised for their po-tential use in biotechnological applications (GALINSKI andTINDALL, 1992; VENTOSA and NIETO, 1995; AGUILAR,1996). Thus extensive studies have been made in recentyears into hypersaline environments, resulting in a largenumber of new halophilic species being isolated.

The family Halomonadaceae included initially twogenera of halophilic bacteria, Halomonas and Chromo-halobacter, and the genus Zymobacter, which comprisednon-halophilic microorganisms (FRANZMANN et al., 1988;DOBSON and FRANZMANN, 1996). Very recently, however,ARAHAL et al. (2002) have proposed that the genus Carni-monas should be included in this family. VREELAND et al.(1980) originally described the genus Halomonas with

only one species, H. elongata, but currently Halomonasincludes 22 species, some of them previously assigned toother genera, such as Arthrobacter (COBET et al., 1970),Deleya (AKAGAWA and YAMASATO, 1989; BAUMANN et al.,1972; BAUMANN et al., 1983; QUESADA et al., 1984;VALDERRAMA et al., 1991) Flavobacterium (VOLCANI,1940), Halovibrio (FENDRICH, 1988), Paracoccus (KOCUR

1984), Pseudomonas (ROSENBERG, 1983; HEBERT andVREELAND, 1987) and Volcaniella (QUESADA et al., 1990).Other species, however, have only been described morerecently as members of the genus Halomonas (FRANZ-MANN et al., 1987; JAMES et al., 1990; BERENDES et al.,1996; ROMANO et al., 1996; MORMILE et al., 1999; DUCK-WORTH et al., 2000; BOUCHOTROCH et al., 2001; YOON etal., 2001; YOON et al., 2002). Halomonas canadensis,Halomonas israelensis, and Halomonas elongata ATCC33174 and DSM 3043 have, on the other hand, been

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Phenotypic characterisation of Halomonas species 361

transferred recently to the genus Chromohalobacter asChromohalobacter canadensis, Chromohalobacter israe-lensis and Chromohalobacter salexigens respectively(ARAHAL et al., 2001a, 2001b).

Our aim has been to describe phenotypically the typestrains of 21 species of the genus Halomonas, studying alarge number of characteristics by means of a reliable,consistent, reproducible approach. For nutritional assayswe used both classical and automated (BIOLOG) testsand therefore our results have allowed us to evaluate theapplication of automated methods to the phenotypicidentification of such bacteria. We have also selectedthose tests which we consider to be most valuable foridentifying new isolates belonging to the genusHalomonas and for differentiating species from eachother. The results of our study should go a long way toclarifying the classification of bacteria included in thisheterogeneous genus.

Materials and Methods

Bacterial strainsWe used the type strains of 21 validly published species be-

longing to the genus Halomonas: Halomonas aquamarina DSM30161T; Halomonas campisalis ATCC 700597T; Halomonas cu-pida CECT 5001T; Halomonas desiderata DSM 9502T;Halomonas elongata CECT 4279T; Halomonas eurihalinaATCC 49336T; Halomonas halmophila ATCC 19717T;Halomonas halodenitrificans CECT 5012T; Halomonas halodu-rans LMG 10144T; Halomonas halophila CCM 3662T;Halomonas magadiensis NCIMB 13595T; Halomonas marinaATCC 25374T; Halomonas marisflavi JCM 10873; Halomonasmaura CECT 5298T; Halomonas meridiana DSM 5425T;Halomonas pacifica ATCC 27122T; Halomonas pantellerensisDSM 9661T; Halomonas salina CECT 5288T; Halomonas sub-glaciescola DSM 4683T; Halomonas variabilis DSM 3051T andHalomonas venusta ATCC 27125T. To determine intraspecificvariation for some selected species we studied 38 strains ofHalomonas halophila (QUESADA et al., 1980); 26 strains ofHalomonas salina (VALDERRAMA et al., 1991); 19 strains ofHalomonas eurihalina (QUESADA et al., 1984) and 4 strains ofHalomonas maura (BOUCHOTROCH et al., 2001).

Maintenance and basal mediumMH medium (QUESADA et al., 1983) with 7.5% (w/v) sea-salt

solution (RODRÍGUEZ-VALERA et al., 1981) was used to maintainthe strains, to prepare the inocula and as the basal medium forphenotypic tests, unless otherwise stated. The pH was adjustedto 7.2 with 1M NaOH except for the media used for growing H.magadiensis and H. campisalis, which were adjusted to pH 9.

Morphological testsGram staining and motility were determined in 24-hour cul-

tures in liquid MH medium. Poly-β-hydroxybutyric acid (PHB)staining was done in 5-day cultures using the method of STANIER

et al. (1966). The morphology and size of colonies and the pro-duction of diffusible and non-diffusible pigments and ex-opolysaccharide were examined in MH medium after 3 days’ in-cubation.

Physiological testsGrowth at different salt concentrations was determined by

spreading 20 µl of each inoculum onto the surface of solid MHmedia with sea-salt concentrations of 0, 0.5, 1, 3, 5, 7.5, 10, 15,

20, 25 and 30% (w/v). The pH growth range was determined ina similar way on MH media in which the pH had been adjustedto 5, 6, 7, 8, 9 or 10 with HCl or NaOH. The temperature rangewas determined as above by incubating the bacteria at tempera-tures of 4, 15, 20, 25, 32, 37 and 45 °C. The ability to growanaerobically was evaluated on solid MH medium incubated injars with the Gas Pak Anaerobic System (BBL).

Biochemical testsOxidase reaction was performed according to KOVACS, 1956.

Catalase was determined by adding 10 volumes of H202 to eachstrain culture (18 h) on solid MH medium.

Acid production from carbohydrates (adonitol, L-arabinose,D-fructose, D-galactose, D-glucose, myo-inositol, lactose, mal-tose, D-mannitol, D-mannose, D-melezitose, L-rhamnose, su-crose, D-salicin, D-sorbitol, sorbose and D-trehalose) was de-tected in liquid MH medium. Doubtful results were checked inLeifson medium (1963) modified for marine bacteria and con-taining large quantities of sugars. The consumption of glucose,either by oxidation or fermentation, was studied in MH mediumto which 1% (w/v) glucose had been added. The medium wasput into Weimberg tubes and the inoculum deposited at the bot-tom of the tube with the aid of a Pasteur pipette; after inocula-tion the tubes were sealed with 2 ml of agar (2% w/v) and 2 mlof paraffin. Respiration in fumarate, nitrate and nitrite wasstudied by the same procedure, the medium being supplementedwith potassium nitrate (2 g/l), potassium nitrite (2 g/l) or sodiumfumarate (0.8 g/l) (CALLIES and MANNHEIM, 1978).

H2S production was tested in liquid MH medium supple-mented with 0.01% (w/v) of L-cysteine; the indicator was a stripof paper impregnated with lead-acetate placed in the neck of thetube (CLARKE, 1953). To assay nitrate reduction 0.2% (w/v)KNO3 was added to the liquid MH medium. Nitrite was detect-ed with naphthylamine/sulphanilic acid reagents and residual ni-trate with zinc dust; gas production was detected in invertedDurham tubes (SKERMAN, 1967).

Gelatine hydrolysis was tested by flooding cultures on solidMH medium containing 1% (w/v) gelatine, with the Frazierreagent (1926). Casein hydrolysis was indicated by a clear zonearound bacterial growth on double-strength solid MH mediumplus an equal quantity of skimmed milk (BARROW and FELTHAM,1993); negative results were confirmed using the Frazierreagent. Starch hydrolysis was tested by flooding cultures onsolid MH medium containing 1% (w/v) starch with Lugol’s io-dine (BARROW and FELTHAM, 1993). Hydrolysis of Tween 20and Tween 80 was tested on solid MH medium supplementedwith 1% (w/v) of separately autoclaved Tween 20 and Tween80 (SIERRA, 1957). Phosphatase production was tested on solidMH medium with 10 ml/l of a filtered, sterilised solution ofsodium phenolphthalein diphosphate (1%) by exposure to aconcentrated ammonia solution (BAIRD-PARKER, 1963). DNAseactivity was revealed on DNA agar supplemented with 7.5%(w/v) sea-salt solution (RODRÍGUEZ-VALERA et al., 1981) by aclear zone after flooding with 1N HCl (JEFFREIS et al., 1957).Haemolysis was studied in solid MH medium supplementedwith 5% (v/v) lamb’s blood. Growth on MacConkey or Cetrim-ide-agar was assessed on these media prepared with 7.5 %(w/v) sea-salt solution. The lecithovitellin test was carried outaccording to LARPENT and LARPENT-GOURGAND (1957) usingsolid MH medium. Tyrosine hydrolysis was revealed by clearzones in cultures on solid MH medium containing 5 g/l tyrosine(GORDON and SMITH, 1955); pigment production was also de-tected in this medium. Aesculin hydrolysis was determined byadding 0.1% (w/v) aesculin and 0.05% (w/v) ferric citrate toMH medium (BARROW and FELTHAM, 1993).

Selenite reduction was determined according to LAPAGE andBASCOMB (1968) with a 4 g/l concentration of sodium hydrogen

362 J. A. MATA et al.

selenite. Gluconate oxidation was tested according to SHAW andCLARKE (1955). The ONPG test was carried out by adding 250ml of a solution containing 6 g/l O-nitrophenyl-β-D-galactopy-ranoside in 0.01 M Na2HPO4 to 750 ml of liquid MH medium(LOWE, 1962). Indol production was tested in liquid MH medi-um using Kovacs’ reagent (1928). Methyl red and Voges-Proskauer were tested using methyl red and Barrit’s reagent(1936) respectively. Urea hydrolysis was detected with Chris-tensen’s medium (1946) and phenylalanine deamination was as-sessed on phenylalanine agar (Difco), both media prepared with7.5% (w/v) sea-salt solution (RODRÍGUEZ-VALERA et al., 1981).Lysine and ornithine decarboxylases were tested according toBARROW and FELTHAM (1993).

Nutritional testsNutritional assays were made using both classical and minia-

turized automated methods. For the former we used Kosermedium (1923) as modified by VENTOSA et al. (1982), (%, w/v):NaCl, 7.5; KCl, 0.2; MgSO4.7H2O, 0.02; KNO3, 0.1;(NH4)2HPO4, 0.1; and KH2PO4. 0.05 filter-sterilised substrate(0.1% w/v) was added to this medium, with the exception ofcarbohydrates, which were used at 0.2% (w/v). When aminoacids were used as substrates the basal medium contained nei-ther KNO3 nor (NH4)2HPO4. The organic compounds used assole source of carbon and energy or sole source of carbon, nitro-gen and energy were the following: aesculin, L-arabinose, D-cel-lobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannose, D-melezitose, D-raffinose, L-rhamnose, ribose, D-salicin, starch, D-trehalose, D-xylose; adonitol, ethanol, glyc-erol, myo-inositol, D-mannitol, sorbitol; acetate, citrate, for-mate, fumarate, gluconate, malonate, propionate, succinate; L-alanine, L-cysteine, L-histidine, DL-isoleucine, L-lysine, L-me-thionine, L-serine and L-valine.

Nutritional tests on 95 substrates were also performed withthe BIOLOG GN2 automated identification system (Hayward,California). Strains were grown on solid MH medium contain-ing 7.5 % (w/v) total salts at 32 °C for 24 h and then suspendedin a sterile solution of 7.5 % (w/v) salts. Cell density was adjust-ed to an OD590 of between 0.34 and 0.39. The BIOLOG GN2microplates were immediately inoculated with 125 µl of cell sus-pension per well and incubated at 32 °C for 24 h. Substrate oxi-dation was measured with a microplate reader at 590 nm.

Antimicrobial susceptibility testsAntimicrobial susceptibility was assayed using the diffusion

agar method (BAUER et al., 1966). The antimicrobial com-pounds (BBL) were: amoxicillin 25 µg, ampicillin 10 µg, carbeni-cillin 100 µg, cefoxitin 30 µg, cefotaxime 30 µg, chlorampheni-col 30 µg, erythromycin 15 µg, kanamycin 30 µg, nalidixic acid30 µg, nitrofurantoin 300 µg, polymyxin B 300 UI, rifampycin30 µg, streptomycin 10 µg, sulphamide 250µg, tobramycin 10µgand trimetroprim-sulphametoxazol 1.25 µg–23.75 µg.

Numerical analysesDifferential characteristics of type strains were coded in binary

form; positive and negative results were coded as 1 and 0 respec-tively; non-comparable or missing characteristics were coded as9. Data were submitted to cluster analysis using a simple match-ing coefficient (SSM) (SOKAL and MICHENER, 1958) and clusteringwas achieved by the unweighted-pair-group method of associa-tion (UPGMA) (SNEATH and SOKAL, 1973). The TAXAN program(Information Resources Group, Maryland Biotechnology Insti-tute, University of Maryland, College Park, HD20742, USA) wasused for computer analysis. Test error was evaluated by examin-ing 10 strains in duplicate (SNEATH and JOHNSON, 1972).

Determination of test scoresTo determine which tests were the most diagnostically con-

clusive in the numerical analysis for differentiating betweenphena we applied Sneath’s formula (1980):

Score =

where PiA, PiB and PiC are the probabilities of character i, inphena A, B and C, and q the number of taxa. The tests withscores higher than 0.5 were considered to have most value fordifferentiating between the strains. With these tests we made asecond numerical analysis using the same methodology as thatdescribed above.

Chi-squared analysis and contingency coefficientTo determine to what extent the phenotypic description of a

given type species represents all the strains included in the specieswe performed a chi-squared analysis and the contingency coeffi-cient (Milton 1994) on four selected species previously describedby us: H. halophila, H. eurihalina, H. salina and H. maura.

Results

The type strains of 21 species belonging to the genusHalomonas were submitted to extensive morphological,physiological, biochemical, nutritional and antimicrobialsusceptibility tests, using the same growth conditions forall the strains, except for H. magadiensis and H. camp-isalis, as we have indicated above. These conditions werechosen as being suitable for the growth of the wide rangeof species in question. We also incorporated into ourstudy other strains of H. halophila, H. eurihalina, H. sali-na and H. maura to make intraspecific comparisons.

The results of the numerical analyses are shown in Fig-ures 1 and 2. The estimated test error was less than 3% ineither case, which would not affect the cluster analysis sig-nificantly. No test was found to be so irreproducible (testvariance ≥ 0.2) as to be excluded from the data matrix.Nevertheless, we did not use the following parameters:colony size and appearance, rod-shape and size, and opti-mum salt concentration for growth due to their beingsomewhat subjective and difficult to evaluate. The tests re-sponsible for most errors were those referring to lysine andornithine decarboxylases, gluconate oxidation and acidproduction from L-arabinose, L-rhamnose and sorbose.

Figure 1 contains the numerical analysis carried outusing 205 differential phenotypic data of type strains(Table 1). At a 62% similarity level most of the typestrains were grouped into three phena (A, B and C), themain difference between them being the capacity of thebacteria included within phenon A to produce acids fromall the sugars tested. Halomonas campisalis, Halomonasdesiderata and Halomonas subglaciescola formed a sepa-rate cluster (phenon C) due to their using few substrateswhen tested with either classical nutritional methods orthe BIOLOG GN2 system. H. campisalis and H. desider-ata showed high similarity (more than 88%) betweeneach other. H. marisflavi did not cluster with the rest ofthe strains at this 62% similarity level.

Phenotypic characterisation of Halomonas species 363

Figure 2 contains the numerical analysis carried outusing the 57 most conclusive diagnostic tests (those withscores of 0.5 or above; Table 2). At a similarity level of62%, we obtained the same clustering as in Figure 1, al-though some strains changed their position slightly in re-lation to the other species. For example, H. marisflavi be-comes more closely related to the species included in phe-non A because it is able to produce acids from sugars. Onthe other hand, H. eurihalina, H. variabilis, H. venustaand H. magadiensis appear more closely related to eachother.

All the strains studied were Gram-negative rods, pro-duced catalase, reduced selenite, and grew at pH valuesof between 8 and 10; at sea-salt concentrations of be-tween 3% and 15% (w/v) and at temperatures between20 °C and 37 °C. Colonies on MH medium containing7.5% (w/v) salts were circular, convex and slightlyopaque with completely smooth edges. They were 2 to 3mm in diameter after 72 h at 32 °C, except those fromEPS-producing bacteria, which formed larger colonies(more than 5 mm). They tested negative for the follow-ing: diffusible pigment, Indol, methyl red, Voges-Proskauer, respiration on fumarate, growth with L-cys-

teine. α-OH-butyric acid, α-keto-butyric acid, and α-keto-valeric acid from BIOLOG GN2 were also negativefor all strains.

Most of the type strains of Halomonas species were ca-pable of using numerous carbon (or carbon and nitrogen)sources. Twenty-one compounds of the BIOLOG GN sys-tem were metabolised by more than 70% of the strains.

As we have mentioned above, Table 1 shows the differ-ential characteristics of type strains of Halomonas speciesordered according to the groups shown in Figure 1. Inthis table we refer to assays never made elsewhere, andalso to those results which do not agree with previouslypublished data. The variations in the phenotypic featuresof the different strains of H. eurihalina, H. halophila, H.salina and H. maura are indicated as percentages ofstrains that give the same responses as the type strains.The results of the chi-squared test and contingency coeffi-cient were 0.6746, 0.6727, 0.6695 and 0.6699 respec-tively.

We also point out those phenotypic features that clear-ly distinguish species from each other (Table 3), amongwhich we indicate some single characteristics that differ-entiate between given species, such as PHB accumulation,

Fig. 1. Dendrogram showing the clustering ofthe type strains of 21 species of Halomonas onthe basis of 205 phenotypic characteristics (95studied by the BIOLOG System). The simplematching (SSM) coefficient and unweighted-pair-group method with average (UPGMA) cluster-ing were used.

364 J. A. MATA et al.

which separates H. halodurans from the other species,phenylalanine deaminase, which was only positive forHalomonas desiderata, and casein hydrolysis forHalomonas halodurans. Halomonas marina was the onlyspecies resistant to rifampycin. As far as Halomonaselongata is concerned, it can be clearly differentiated byits capacity to metabolise sebacid acid. Finally,Halomonas marisflavi could easily be distinguished fromthe other species because of three features: haemolysis(positive), lecithinase (positive) and its resistance totrimetroprim-sulphametoxazol, all of which explains whythis species did not cluster at a 62% similarity level withthe rest of the species.

Discussion

In the course of our taxonomic studies we can verify thatthe majority of halophilic microorganisms isolated from hy-persaline habitats are Gram-negative rods with a respirato-ry metabolism and are related to the genus Halomonas.Nevertheless, the differentiation of Halomonas species onthe basis of their phenotypic characteristics has so far

proved quite difficult for various reasons. In some cases thephenotypic description of a given species has been made onthe basis of a relatively low number of characteristics, as oc-curs for example with Halomonas halodenitrificans(KOCUR, 1984), H. marina (COBET et al., 1970) and severalother species described some years ago. Furthermore, thecharacteristics selected by some authors to describe aspecies might be different from those selected by other au-thors; a case in point, the old Deleya species (BAUMANN etal., 1972; 1983) was described using nutritional data alone.In addition, the methodology followed to determine a givencharacteristic may not always be the same, as for examplewith acid production from sugars. And finally, some au-thors have not described the methods used; for example, H.halmophila was originally described in 1940 as Flavobac-terium halmephilum (VOLCANI, 1940); later, in 1990, DOB-SON et al. added to the description of this microorganism bymeans of nearly a hundred further tests without, unfortu-nately, describing the techniques used.

In the review of halophilic microorganisms publishedby VENTOSA et al. (1998), the authors included a tablesummarising the differential features among Halomonasspecies. For their purpose they relied upon data from

Fig. 2. Dendrogram showing clustering of typestrains of species of Halomonas on the basis of57 phenotypic characteristics with scores of > 0.5.The simple matching (SSM) coefficient and un-weighted-pair-group method with average(UPGMA) clustering were used.

Tab

le 1

.Ph

enot

ypic

cha

ract

eris

tics

of

type

str

ains

of s

peci

es o

f the

gen

us H

alom

onas

.1.

Hal

omon

as a

quam

arin

a; 2

. Hal

omon

as m

erid

iana

; 3. H

alom

onas

cup

ida;

4. H

alom

onas

pan

telle

rien

sis;

5. H

alom

onas

hal

mop

hila

; 6. H

alom

onas

elo

ngat

a; 7

. Hal

omon

as v

aria

bilis

;8.

Hal

omon

as e

urih

alin

a; 9

. Hal

omon

as m

agad

iens

is; 1

0. H

alom

onas

sal

ina;

11.

Hal

omon

as h

alod

enit

rifi

cans

; 12.

Hal

omon

as m

aura

; 13.

Hal

omon

as h

alop

hila

; 14.

Hal

omon

as p

aci-

fica

; 15

. H

alom

onas

ve

nust

a;

16.

Hal

omon

as

halo

dura

ns;

17.

Hal

omon

as

mar

ina;

18

; H

alom

onas

ca

mpi

salis

; 19

. H

alom

onas

de

side

rata

; 20

. H

alom

onas

su

bgla

cies

cola

;21

. Hal

omon

as m

aris

flav

i.

Tab

le 1

.C

onti

nued

.

Tab

le 1

.C

onti

nued

.

Tab

le 1

.C

onti

nued

.

Tab

le 1

.C

onti

nued

.

Dat

a sh

aded

in g

rey

indi

cate

cha

ract

eris

tics

not a

naly

sed

in p

revi

ous

desc

ript

ions

; ast

eris

ks in

dica

te r

esul

ts th

at a

re n

ot in

acc

orda

nce

with

pre

viou

s de

scri

ptio

ns; b

old

char

acte

rist

ics

indi

cate

use

ful

test

s fo

r dis

tingu

ishi

ng a

par

ticul

ar s

peci

es.

a %

of s

trai

ns th

at re

act i

n th

e sa

me

way

as

the

type

str

ain.

b

Whe

n su

pplie

d as

the

sole

sou

rce

of c

arbo

n an

d en

ergy

or a

s th

e so

le s

ourc

e of

car

bon,

nitr

ogen

and

ene

rgy.

Tab

le 2

.Ph

enot

ypic

cha

ract

eris

tics

wit

h sc

ores

of 0

.5 o

r ab

ove

of ty

pe s

trai

ns o

f spe

cies

of t

he g

enus

Hal

omon

as.

1. H

alom

onas

aqu

amar

ina;

2. H

alom

onas

mer

idia

na; 3

. Hal

omon

as c

upid

a; 4

. Hal

omon

as h

alm

ophi

la; 5

. Hal

omon

as p

ante

lleri

ensi

s; 6

. Hal

omon

as m

aris

flav

i; 7.

Hal

omon

as e

long

ata;

8.

Hal

omon

as e

urih

alin

a; 9

. Hal

omon

as v

aria

bilis

; 10.

Hal

omon

as v

enus

ta; 1

1. H

alom

onas

mag

adie

nsis

; 12.

Hal

omon

as h

alod

enit

rifi

cans

; 13.

Hal

omon

as m

aura

; 14.

Hal

omon

as s

alin

a; 1

5.H

alom

onas

hal

odur

ans;

16.

Hal

omon

as m

arin

a ;

1

7. H

alom

onas

hal

ophi

la;

18;

Hal

omon

as p

acif

ica;

19.

Hal

omon

as c

ampi

salis

; 20

. H

alom

onas

des

ider

ata;

21.

Hal

omon

as s

ub-

glac

iesc

ola.

Tab

le 2

.C

onti

nued

.

aW

hen

supp

lied

as th

e so

le s

ourc

e of

car

bon

and

ener

gy.

Tab

le 3

.U

sefu

l tes

ts fo

r di

stin

guis

hing

the

type

str

ains

of s

peci

es o

f the

gen

us H

alom

onas

.1.

Hal

omon

as a

quam

arin

a; 2

. Hal

omon

as m

erid

iana

; 3. H

alom

onas

cup

ida;

4. H

alom

onas

hal

mop

hila

; 5. H

alom

onas

pan

telle

rien

sis;

6. H

alom

onas

mar

isfl

avi;

7. H

alom

onas

elo

nga-

ta; 8

. Hal

omon

as e

urih

alin

a; 9

. Hal

omon

as v

aria

bilis

; 10.

Hal

omon

as v

enus

ta; 1

1. H

alom

onas

mag

adie

nsis

; 12.

Hal

omon

as h

alod

enit

rifi

cans

; 13.

Hal

omon

as m

aura

; 14.

Hal

omon

assa

lina;

15.

Hal

omon

as h

alod

uran

s; 1

6. H

alom

onas

mar

ina;

17.

Hal

omon

as h

alop

hila

; 18;

Hal

omon

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Phenotypic characterisation of Halomonas species 373

original publications of these species and therefore 40%of the species lacked data for more than 30% of the dif-ferential characteristics cited. ARAHAL et al. (2002) haverecently published a phylogenetic study of the Halomon-adaceae family, in which they point out the need for phe-notypic data to be used together with their results in a re-classification of the genus Halomonas.

For all these reasons we have characterised the typestrains of 21 species of Halomonas via 234 phenotypictests. Moreover, we have used the same methods through-out, performing the phenotypic tests under consistent,standard conditions to obtain reproducible results (VAN-DAMME et al., 1996). It is well known for example thatthe salt concentration might modify the results of somephenotypic tests; in this way, H. halodurans has beenshown to suffer some phenotypic changes when the saltconcentration was lowered from 8% to 1.7% (w/v) (HER-BERT and VREELAND, 1987) Thus we chose to carry outour assays at a salt concentration of 7.5 % (w/v), themost appropriate concentration for the growth of thewide range of Halomonas strains we are dealing with.

As can be seen, most of the tests gave the same resultsas those published elsewhere by the authors who original-ly described these strains. Nevertheless, some of our re-sults (indicated with asterisks in Table 1) did not coincidewith the previously published data, although these casesonly amounted to 4% of the total. Most of these differ-ences might be due to the different methods used.

We determined some characteristics by a second tech-nique to confirm doubtful results and those of our resultswhich were not in accordance with data obtained fromthe original reports. For example, to test acid productionfrom sugars, we used two different culture media: Leifsonmedium, with a low peptone and high carbohydrate con-tent (LEIFSON, 1963), and MH basal medium (QUESADA etal., 1983), which had more peptones and a lower carbo-hydrate content. In this way our results indicated that H.elongata was not able to ferment glucose, just as otherauthors such as VENTOSA et al. (1998) have also observed.

We also found some differences in the nutritional testscarried out using classical and automated methods. Thesediscrepancies in the results were probably due to their dif-ferent operational modes: increased growth by assimila-tion of carbon sources (classical method) or oxidation ofsubstrates via an electron transport chain (BIOLOGmethod) (RUGER & KRAMBECK, 1994). So we distinguishedtwo sets of data, substrate use and substrate metabolism,as recommended by RUGER & KRAMBECK (1994).

In the case of Halomonas halodurans we studied twocultures from two different collections, DSM and LMG.At first we worked with H. halodurans DSM 5160T butwe found quite significant differences between our initialresults and the data published by the authors of the firstreport, such as acid production from some sugars, H2Sproduction and aesculin and Tween 80 hydrolysis andthus we decided to use Halomonas halodurans LMG10144T to carry out this study. Other authors have alsoobserved similar discrepancies between strains from dif-ferent sources JAMES et al. (1990).

We subsequently determined which tests gave themost conclusive results in differentiating between the

phena shown in Figure 1 by means of the analysis de-scribed by SNEATH (1980). Using the selected tests (thosewith scores of 0.5 or above, cf. Table 2), we made a sec-ond numerical analysis, from which we obtained thesame clustering of species (Figs 1 and 2). This will allowfuture researchers to reduce the number of phenotypicdata needed to conduct a numerical taxonomic study ofsuch bacteria.

We then went on to make intraspecific comparisonsbetween the type strains of four species described by us,H. halophila (QUESADA et al., 1984), H. eurihalina (QUE-SADA et al., 1990, H salina (VALDERRAMA et al. 1991) andH. maura (BOUCHOTROCH at al., 2001) and other strainsof these species. The chi-squared test and that of the coef-ficient of contingency revealed a high degree of homo-geneity between the type strains and the rest of the strainsclassified within these species, since the contingency coef-ficient was in all cases close to 0.67 in an interval from 0to 0.7. Furthermore, the strains belonging to the samespecies were highly similar to each other, as the resultsshown in Table 1 indicate.

Our work here represents a thorough phenotypic anal-ysis and the first numerical taxonomic study for the typestrains of Halomonas species. To the best of our knowl-edge this is the first time that the BIOLOG system hasbeen applied to this group of halophilic bacteria. The oxi-dation of some substrates included in BIOLOG, togetherwith acid production from sugars, leads us to groupHalomonas species into three distinct phena. Further tothis, enzyme-activity assays and biochemical and physio-logical tests allow us to distinguish quite clearly betweenindividual species.

We believe that the data contained in this reportshould be very useful to other researchers working withhalophilic microorganisms. Firstly, the data included inthe Tables should be of great use to future researchers foridentifying an isolate of Halomonas either as a new taxonor a member of an already described species. Secondly,we have determined how to carry out a reliable numericalanalysis without using an inordinate number of tests.Many of the tests selected for such an analysis can bemade in BIOLOG systems and are therefore easy, reliableand reproducible (Table 2). Finally, we have been able todiscover many essential phenotypic characteristics thatdistinguish Halomonas species from each other (Table 3).These characteristics can be determined by tests whichare relatively simple but at the same time are highly effi-cient for species differentiation. The tests reveal physio-logical, biochemical and nutritional features as well asantimicrobial susceptibility.

Our study also provides the phenotypic data neccesaryto complement phylogenetic studies already carried outby other authors working in the field, since it is becomingclear that the taxonomic position of some of species ofthe genus Halomonas should be reconsidered (ARAHAL etal., 2002).

AcknowledgementsThe authors thank A. VENTOSA and D. R. ARAHAL for

supplying some of the strains and A. VENTOSA for his crit-

374 J. A. MATA et al.

ical reading of our manuscript and valuable discussions.This research was supported by a grant from the Direc-ción General de Investigación Científica y Técnica(BOS2000-1519) and from the Plan Andaluz de Investi-gación, Spain. We also thank A. L. TATE for revising theEnglish text and M. VALDERRAMA for helping us with thestatistical interpretation of our data.

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Corresponding author:Dr. E. QUESADA, Dpto. de Microbiología, Facultad de Farmacia,Campus Universitario de Cartuja s/n., 18071 Granada, SpainTel.: ++34-958-243871; Fax: ++34-958-246235;e-mail: equesada@ugr.es